Non-secretory renin reduces oxidative stress and increases cardiomyoblast survival during glucose and oxygen deprivation

Figures

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  Figure 1. Expression of renin transcripts in transfected pIRES and ren(2-9) cells. Cardiac pIRES controls (empty vector-transfected cells) and ren(2-9)-overexpressing cells [Ren(2-9)] were exposed to control conditions, glucose (Glc) depletion alone, anoxia alone, or the combination of oxygen and glucose depletion (OGD) for 24 hours. (A–D) Renin transcript levels or (F) hypoxia-relevant genes normalized to the housekeeper YWHAZ were quantified by RT-PCR. (E) Renin protein of pIRES controls and ren(2-9) cells normalized to the protein content was detected by Western Blot. The data represent mean ± SEM values of 5-7 independent experiments or representative Western blots. *p < 0.05, ***p < 0.001 (Anova, Kruskis-Wallis (A–D) or Bonferroni (E)), E(1A-9): exon(1A-9)renin, E(1-9): exon(1-9)renin.

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  Figure 2. Ren(2-9) protects H9c2 cells from apoptotic and necrotic death induced by ischemia-related conditions. Cardiac H9c2 pIRES controls (empty vector-transfected cells) and ren(2-9)-overexpressing cells [Ren(2-9)] were exposed to control conditions, glucose (Glc) depletion alone, anoxia alone, or the combination of oxygen and glucose depletion (OGD) for 24 hours. (A) Representative histograms of apoptotic cells labelled with apoptosis-specific markers and propidium iodide (PI). Apoptosis rate was quantified by the percentage of (B) CaspACE+, (C) Annexin V+ and (D) Fas receptor+ cells (n = 8–9, each). Cells were additionally labelled with PI to differentiate between early apoptosis (PI−) and late apoptosis (PI+) (grey shaded). Necrosis rate was determined (E) by PI labelling (early necrosis, PI+ apoptosis- cells, n = 9) and (F) by the percentage ratio between released LDH and LDH content using the cytotoxicity detection kit (n = 10). Data represent mean ± SEM values. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. basal conditions with glucose and oxygen; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. pIRES controls.

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  Figure 3. Ren(2-9) protects H9c2 cells from accumulation of mitochondrial superoxides induced by ischemia-related conditions. Cardiac pIRES controls (empty vector-transfected cells) and ren(2-9)-overexpressing cells [Ren(2-9)] were exposed to control conditions, glucose (Glc) depletion alone, anoxia alone or the combination of oxygen and glucose deprivation (OGD) for 24 hours. Afterwards, cells were incubated with MitoSOX fluorophore to detect mitochondrially localized superoxides. (A) Representative histograms and (B) analyses of percentage and mean fluorescence intensity of MitoSOX+ cells. Data represent mean ± SEM values of 9-10 experiments. ***p < 0.001 vs. basal conditions with glucose and oxygen; #p < 0.05, ##p < 0.01 vs. pIRES controls.

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  Figure 4. Ren(2-9) protects H9c2 cells from accumulation of cytosolic reactive oxygen species induced by ischemia-related conditions. Cardiac pIRES controls (empty vector-transfected cells) and ren(2-9)-overexpressing cells [Ren(2-9)] were exposed to control conditions, glucose (Glc) depletion alone, anoxia alone or the combination of oxygen and glucose deprivation (OGD) for 24 hours. Afterwards, cells were incubated with dihydroethidium (DHE) to detect reactive oxygen species in the cytosol. (A) Representative histograms and (B) analyses of percentage (left panel) and mean fluorescence intensity of DHE+ cells (right panel). Data represent mean ± SEM values of 7-10 experiments. ***p < 0.001 vs. basal control with glucose and oxygen; ###p < 0.001 vs. pIRES cells.

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  Figure 5. Ren(2-9) protects H9c2 cells from disruption of mitochondrial membrane potential induced by ischemia-related conditions. Cardiac pIRES controls (empty vector-transfected cells) and ren(2-9)-overexpressing cells [Ren(2-9)] were exposed to control conditions, glucose (Glc) depletion alone, anoxia alone or the combination of oxygen and glucose deprivation (OGD) for 24 hours. Afterwards, cells were incubated with JC1 dye to detect the mitochondrial membrane potential (∆Ψm). (A) Representative histograms of JC1+ cells separated in different quadrants according to the generation of JC1 aggregates (red fluorescence) or JC1 monomers (green fluorescence). (B) Analysis of the ratio of red/green fluorescence intensity (FLI) representing the ∆Ψm. (C) Distribution of JC1+ cells within the different quadrants according to their red or green fluorescence. Localization of quadrant 1: upper left, quadrant 2: upper right, quadrant 4: bottom right. Data show mean ± SEM values of 8 experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. pIRES cells, #p < 0.05, ##p < 0.01, ###p < 0.001 vs. basal controls.

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  Figure 6. Ren(2-9) protects H9c2 cells from mitochondrial calcein overload induced by ischemia-related conditions. Cardiac pIRES controls (empty vector-transfected cells) and ren(2-9)-overexpressing cells [Ren(2-9)] were exposed to control conditions, glucose (Glc) depletion alone, anoxia alone or the combination of oxygen and glucose deprivation (OGD) for 24 hours. Afterwards, cells were incubated with calcein-AM or calcein-AM plus CoCl2 to monitor the distribution of calcein within all subcellular compartments combined vs. mitochondria alone. (A) Representative histograms of total vs. mitochondrial calcein fluorescence intensities (FLI). (B) Analyses of total (white columns) vs. mitochondrial calcein FLI (grey columns). (C) Ratio of mitochondrial to total calcein FLI representing mitochondrial permeability transition. Data represent mean ± SEM values of 8 experiments. *p < 0.05, **p < 0.01 vs. pIRES cells, ##p < 0.01, ###p < 0.001 vs. basal control.

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  Figure 7. Ren(2-9) protects H9c2 cells from OGD-induced cytosolic Ca2+ overload. Cardiac pIRES controls (empty vector-transfected cells) and ren(2-9)-overexpressing cells [Ren(2-9)] were exposed to control conditions, glucose depletion, anoxia or the combination of oxygen and glucose deprivation (OGD) for 24 hours. Afterwards, cells were incubated with the fluorophore Fluo3AM to detect cytosolic free Ca2+ levels via analysis of fluorescence intensity. Data show mean ± SEM values of 10 experiments. **p < 0.01 basal vs. control, ##p < 0.01 vs. pIRES cells.

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  Figure 8. Ren(2-9) limits decrease of ATP levels induced by ischemia-related conditions. Cardiac pIRES controls (empty vector transfected cells) and cyto-renin overexpressing cells [Ren(2-9)] were exposed to control conditions, glucose depletion alone, anoxia alone or the combination of glucose and oxygen deprivation (OGD) for 24 hours. Afterwards, cellular ATP levels were determined by the CellTiter Glo Cell Viability Assay. Data show mean ± SEM values of 6 experiments. ***p < 0.001 vs. basal control, ##p < 0.01, ###p < 0.001 vs. pIRES.